ELISA: General guidelines and

troubleshootings

Dr Rosy Sultana MBBS,Mphil

Professor (cc) & Head
Dept of Immunology
Bangladesh University of
Health Sciences (BUHS)
ELISA

Enzyme
Linked
ImmunoSorbent
Assay
 What is ELISA?
 Is a popular format of a “wet-lab” type analytical
assay that uses a solid –phase enzyme immunoassay to
detect the presence of an antigen or an antibody in a
liquid sample
 Immunological technique: uses antibodies
 Qualitative and Quantitative
 Very sensitive (0.0001 – 0.001 µg/ml)
 Commonly used in medicine and scientific research
 History of ELISA
 Radioimmunoassay by R Yalow and S Berson in 1960
 Antibody linking to enzyme by S Avrameas and GB Pierce
 Imunosorbent assay by Wide and Porath in 1966
 P Perlmann and E Engvall from Sweden and, A Schuurs
and B van Weemen from Netherlands independently
synthesized their knowledge into methods to perform
ELISA in 1971
 Basis of ELISA

ELISA is based on specific binding between Antibodies and Antigens

 Heavy
chain

Disulfide Light
bonds chain

ELISA
Antibody
Structure
 Basic steps of ELISA

 Antigen (solid-phase) is absorbed on to plastic surface

(sorbent).
 Antigen is recognised by specific antibody (immuno).
 This antibody is recognised by second antibody
(immuno) which has enzyme attached (enzyme-linked).
 Substrate reacts with enzyme to produce product,
usually coloured.

Coloured product = measure (assay) of

antigen present
 Secondary
antibody
Substrate
ym e
Enz
Coloured
product

Primary
antibody

Coated antigens in Microtiter well

General Types of ELISA
 Qualitative ELISA
 Postive or Negative results

 Quantitative ELISA
 Optical density or fluorescent units of the sample is
interpolated into a standard curve, which is
typically a serial dilution of the target.

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 General principles of ELISA

 A Polystyrene microtiter plate is coated with a

constant amount of Ab/Ag
 Patient’s serum is then added which may/may not
contain the Ag/Ab of interest
 Ag/Ab if present in patient’s serum binds to the
immobilized Ab/Ag in the plate during incubation
 The plate is then incubated with an enzyme –labeled
antibody (conjugate)
 General principles of ELISA (Cont’d)
 The conjugates react with the Ag-Ab complex on the
plate
 Between each step the plate is typically washed with a
mild detergent solution (wash buffer) to remove
unbound proteins or antibodies that are not specific.
 After final wash step the plate is developed by adding
an enzymatic substrate to generate a visible signal, a
colored reaction product that is measured by a
Spectrophotometric plate reader.
General guidelines- diagrammatic overview
 Antibodies in ELISA
 Generally, monoclonal antibodies are used because
they are more specific for a certain epitope than
polyclonal antibodies.
 They are originally produced by inducing a humoral
immune response in an animal (eg. Rat or mouse) by
injecting an antigen many times, then cells are
extracted and antibodies are purified.
 They can also be extracted from humans who have
been immunized to a certain antigen
 Enzyme Function
 Enzymes are proteins that speed up the rate of a
chemical reaction without being used up and usually
react only to particular substrates.
 The rate of this reaction is proportional to the
amount of enzyme present.
 In the case of non-competitive ELISA, the more
binding of the enzyme conjugate to the antigen, the
stronger will be the color development.
 Mechanism of enzyme action

 React with a colourless substrate to produce a coloured

product.
 Must work fast at room temperature so the colour
develops quickly.
 Have minimal interference from factors in sample.

Peroxidase from horseradish

Alkaline phosphatase from E coli
-galactosidase from E coli
 Enzymes used in ELISA

 Horse-radish peroxidase (HRP)

 Alkaline phosphatase
 -galactosidase
 Streptavidin
 Substrate Function

 A substrate is a compound or substance that

undergoes change.
 Substrates bind to active sites on the surface of
enzymes and are converted or changed.
 In ELISA the specific substrate used changes color.
 Substrates used in ELISA

 Hydrogen peroxide
 3,3´, 5,5´ Tetra methyl benzidine (TMB)
 p-nitrophenyl phosphate (pNPP)
 Bromochloro-Indolyl phosphate (BCIP)
An ELISA Laboratory needs following
items:

Microplate reader
•

Printer
•

Microplate incubator
•

Plate wash system

•

Record Sheet
•

Dilution Tubes
•

Micropipettes and pipette tips

•
Microplate Incubator
Microwell Plate Washer
Microplate (ELISA) Reader
Materials Needed

Contents of a ELISA Kit

1. ELISA plate
2. Positive control
3. Negative control
4. Dilution Buffer
5. Wash Buffer (PBS, Tween 20)
5. Conjugate (Enzyme linked secondary antibody)
6. TMB Substrate
7. Stop solution
 Types of ELISA
 Indirect ELISA
 Antigen bound to plate
 Detects antibodies
 Sandwich ELISA
 Antibody bound to plate
 Detects antigens
 Competitive ELISA
 Antibody /antigen bound to plate
 Detects antibodies or antigen
(Depends on type of conjugate used)
 i

Indirect ELISA
Sample added to Microtiter
1st step well (Ag coated)
Incubation
↓
Wash
2nd step ↓
Conjugate (enzyme labeled
anti-isotype Ab) added
↓ Incubation
Wash
3rd step ↓
Addition of substrate
↓ Incubation
Color development
↓
Stop solution added
↓
OD measured
Indirect ELISA
 Sandwich ELISA
Sample added to Microtiter well
1st step (monoclonal Ab coated)
↓ Incubation
Wash
↓
2nd step Conjugate (enzyme labeled
polycloncal Ab) added
↓ Incubation
Wash
3rd step ↓
Addition of substrate
↓ Incubation
Color development
↓
Stop solution added
↓
OD measured
Sandwich ELISA

 The antibodies fixed to a

solid surface, such as the
inner surface of a test tube;

 A preparation of the same

antibodies coupled to an
enzyme.

 This is one enzyme that

produces a colored product
from a colorless substrate.

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 Competitive ELISA
Serum and recombinant Ag added
1st step to microwell plate Incubation
(monoclonal Ab coated)

Wash
2nd step 
Conjugate added Incubation
(enzyme labeled human Ab to Ag)

Wash

3rd step
A substrate added Incubation

Stop solution added

OD measured

No color indicates positive result, color indicates negative result

Competitive ELISA
 Microplate reader
 Machine which measures color
density in plate well.
 A specific wave-length of light is
chosen (depending upon the color
you expect to capture).
 Light is cast from the bottom of
the well through the sample.
 No color change- wells absorb very
little light;
 Wells with color change absorb
more light; this is the optical
density or OD.
 Reading plates
 Select proper wave-length on machine.
 Carefully wipe bottom of plate to remove
excess moisture. Plates that are wet may
diffuse your light source, giving inaccurate
readings.
 Mix the plate. This will distribute color
evenly. Some machines have a ‘mix’ setting.
 ELISA results
Results should be recorded by reading the optical
densities of the plates in a plate reader at the correct
wavelength:
IDEXX: 450nm
Each manufacturer supplies computer software specific
for their test which calculates which samples are
negative and the titers of positive samples.
ELISA Kit
Results
 Calculation
 The result of a typical test is calculated by comparing
the spectrophotometric reading of unknown serum to
that of reference serum.
 Average the 2 negative control wells
 Average the 2 positive control wells
 Average 2 wells for each sample
 The intensity of the color is directly proportional to
the amount of Ag/Ab present in patient’s sample in
Indirect and Sandwich ELISA but inversely
proportional in case of Competitive ELISA.
Calculation of results
Calculation of results, cont’d
 Results
The status of a sample are evaluated by the
sample to positive ratio (S/P ratio):

Sample mean - negative control mean

positive control mean - negative control mean

(mean of optical absorbance)

With the IDEXX kit S/P ratios of greater than

0.5 are considered positive
 Calculation of Results
Example:
Sample mean= 0.820
Negative control mean=0.053
Positive control mean=0.563

0.820 - 0.053 = 0.767

= 1.5 or Positive
0.563 - 0.053 = 0.510

S/P ratios of greater than 0.5 are considered positive

(Positive values will be different for each kit)
The ELISA assay yield three different types of data output

 Qualitative: ELISA can be used to achieve a YES or NO

answer indicating whether a particular antigen is present in a
sample, as compared to a blank well containing no antigen or
an unrelated control antigen.
 Quantitative: ELISA data also can be interpreted in
comparison to a standard curve in order to precisely calculate
the concentrations of antigen in various samples.
 Semi-quantitative: ELISA can be used to compare the
relative levels of antigen in assay samples, since the intensity of
signal will vary directly with antigen concentration.
 Run validation criteria for controls
Negative control absorbance- > 0.010 or <0.050
The difference between the positive and negative
control mean absorbance must be ≥ 0.50
Example: if negative control - 0.030, the positive
control must be 0.530 or greater.
Precision is important for an ELISA
 Advantages of ELISA
 Reasonably sensitive –possible to detect less than a
nanogram of protein.
 No radiation hazards.
 Reagents are cheap with long shelf life.
 Handles large number of samples at a time.
 Simple technique –color change.
 Adaptable to automation and high speed.
 Qualitative and quantitative
 Detection kits available commercially
Why ELISA is considered as a reliable
and precise technique in immunoassay?

 Good opportunity to use controls/ standards in the

assay all times
 Provides highly consistent and reproducible test
result - random variability is absent
 An acceptable cut off range determines negative
and positive results
 Reliability of the assay always depends on its run
validation criteria
 Disadvantages of ELISA
 Time consuming test
 Generally requires incubation
-can not take out into the field
 Can only test for a specific antibody or antigen
 Highly skilled technician is required for quality test
 False positive- if wash is improper and inadequate
 Application of ELISA
Types of
Protocol Application
ELISA
Tracking
HIV, SARS, Dengue, Bird-flu,
I outbreaks of
anthrax, Ebola & Zika
disease

Detecting HBV, HDV, Pregnancy, drugs

II
antigens (including abusive drugs)

Detecting
HAV, HBV, HCV, HDV, HEV,
III antibodies in
HIV, H. pylori, TB, Dengue
serum
 Application of ELISA…. cont’d
Protocol Types of ELISA Application

Screening of
HIV-1,HIV-2(Presence of anti-HIV
blood for
IV Abs), Hepatitis B (for both Ag & Abs),
evidence of viral
Hepatitis C (presence ofAbs)
contamination

HCG (as a test for pregnancy), LH

Measuring
V (determining the time of ovulation),
hormone levels
TSH, T3 and T4 (for thyroid function)

STD like HIV, syphilis and chlamydia

Detecting
VI Hepatitis B and C
infections
Toxoplasma gondii
 Application of ELISA (Cont’d)

 Detection of allergens in food, eg. milk, peanuts,

walnuts, almonds, eggs.

 Detection of illegal drugs in Sports Medicine.

If negative controls are giving positive results

 Contamination of the substrate solution, enzyme labeled

antibody or control themselves

 Inadequate rinsing of plates

 Inadequate blocking of plates

If no color develops for the positive controls or
for the samples

 Check all reagents for expiry dates and storage condition

 Microwell plates may not be coated properly

 Reagents applied in wrong order or an assay step was
omitted
 Wrong conjugate was used- conjugate was prepared
incorrectly or has deteriorated
 Incorrect or no detection antibody was added
 Substrate solution was not added
If very little color develops for positive controls
and the test samples

 Laboratory temperature was too low or reagents/plates

were too cold
 Check the dilution of the enzyme labeled antibody and
concentration of the substrate
 Wash buffer not adequately drained after every wash step
or too many wash cycles
 Incubation periods were too short
 Wrong conjugate was used- conjugate was prepared
incorrectly or has deteriorated
ELISA Troubleshooting- poor Reproducibility

Causes:

 Excessive time was taken to add samples, controls, or

reagents to the assay plate
 Multichannel pipette was not functioning properly
 There was inconsistent washing or washer system
malfunctioning
 There was poor distribution of antibody in the sample
If color develops for the test samples but not for
the positive controls

 Check the source of positive controls, their expiry

date and storage condition
If the color can be seen, but the absorbance is
not high as expected

 Check the wave length of filter.

Thank you